Microscope with light sheet illumination

Abstract

A family of microscopes include an illumination device which produces a planar light sheet along an illumination axis of an illumination beam path and a transverse axis normal to the illumination axis. A detection device detects light emitted from the sample region along an axis of detection of a detection beam path. The illumination and detection axes as well as the transverse axis and the axis of detection being oriented relative each other at an angle unequal to zero. A light sheet generator also produces rotationally symmetrical light and includes structure and control for rapidly scanning the sample region along the transverse axis. The illumination device includes a second light sheet generator having a first astigmatically active optical element with at least one astigmatic lens for producing a static sheet of light. Selection elements used to select either the first or the second light sheet or both together to produce the sheet of light.

Description

PRIORITY CLAIM[0001]The present application is a National Phase entry of PCT Application No. PCT / EP2010 / 063666, filed Sep. 17, 2010, which claims priority from German Application Number 102009044984.1, filed Sep. 24, 2009, the disclosures of which are hereby incorporated by reference herein in their entirety.FIELD OF THE INVENTION[0002]The invention relates to a microscope comprising an illumination device which produces a light sheet to illuminate a sample region, the said sheet having an approximately planar extension in the direction of an illumination axis X of an illumination beam path and in the direction of a transverse axis Y lying across the illumination axis X. The microscope further comprises a detection device used to detect light that is radiated by the sample region along a detection axis Z of a detection beam path, the illumination axis X and the detection axis Z as well as the transverse axis Y and the detection axis Z being oriented relative to each other at an angl...

AI Technical Summary

Benefits of technology

[0024]The angle scanning mirror may be arranged conjugated to the illumination focal plane if the second means for light sheet generation have been selected, i.e., if, for example, the cylindrical optical element is in the beam path. By means of the angle scanning mirror, the sample can be illuminated from different angles, which can be used to reduce banding.

Problems solved by technology

All the setups and methods known in prior art, however, have more or less grave disadvantages, which restrict the use of the SPIM technology in the commercial sphere, where it is important, among other things, to achieve a high user friendliness of the microscopes and, as a rule, a high throughput, with a great number of samples having to be examined within a relatively short time.

In most of the setups using SPIM technology that have been implemented so far, e.g., those according to DE 102 57 423 A1 and WO2004 / 053558A1, the mere variation of the image field size for detection—e.g., switching from an image field size providing a good overview of the sample to a detail region—is rather a complex and time-consuming affair.

This affects the sample space unfavorably, which may have a particularly negative effect in case of a horizontal detection beam path.

Moreover, the sample is unnecessarily heated or cooled.

Nevertheless, slight interactions with the sample chamber and, thus, indirectly with the sample cannot be avoided.

This, too, is inserted into the sample chamber from above, which is, as a rule, filled with an immersion liquid, so that, here again, there are slight interactions with the sample chamber when the zoom function of the objective is working or when the focus is adjusted, alone because of the motorized shifting of the lenses, which may cause vibrations that may transmit to the liquid in the sample chamber.

The flare occurring in case of diaphragms causes light losses, whereas the use of beam expanders reduces flexibility, since exchanging them is rather laborious.

If plain scanning is combined with angular scanning, i.e. illumination from different angles, in order to reduce banding as described, e.g., in DE 10 2007 015 063 A1, there is a risk that beat artefacts will be produced if the scanners for light sheet angle scanning and position scanning are not matched, i.e. not synchronized.

For many kinds of samples, such as embryos of the fruit fly (Drosophila), such a setup is not of advantage, because in this way scattering and non-scattering image portions are combined in an unfavorable way.

This applies especially to horizontal detection beam paths with immersed detection objectives: Here, the necessary movement of the objective entails tightness problems.

On the other hand, a movable element in the sample space is disturbing in general, as the user may need space there for diverse means for feeding to the sample chamber.

The vibrations occurring during the movement of the objective may be unfavorably transmitted to the sample, since the space between the objective and the sample is occupied by a liquid rather than by air.

If scanners are used for producing the light sheet, the imaging of the scanner into the pupil of the illumination objective is not optimal, as a rule, so that the plain position scanning is superposed by portions of angular scanning.

In the direction of the reflected light, however, image artefacts such as astigmatism or also defocusing do occur, due to surface tensions at the dichroic filter, which can be caused, e.g., by the coating, or by improper installation.

As far as the reflected branch beam path is concerned, though, the problem of surface tensions may occur here, too, if conventional dichroic filters are used.

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